Directed evolution techniques for engineering proteins in vitro combine random mutagenesis with screening assays that permit rapid selection of cells expressing protein variants with the desired characteristics. Directed evolution technology is not limited by the availability of structural information and is broadly applicable, as demonstrated by its use in the successful engineering of enzymes and antibodies in bacterial expression systems. However, to date, directed evolution has not been used in mammalian expression systems because limitations associated with the transformation of mammalian cells have prevented the efficient expression of large protein libraries in which each cell expresses a single gene variant. In this proposal, site-specific integration of DNA mediated by Cre- recombinase will be used to establish a general system for the expression of protein libraries in mammalian cells. To demonstrate the utility of Cre- mediated targeted insertion for protein engineering in mammalian cells, combinatorial protein libraries of the bleomycin resistance protein (BRP) will be expressed, sequenced, and screened. Expression of a diverse BRP library In mammalian cells coupled with the successful application of directed evolution to modify the functional characteristics of BRP provides a model system for engineering other potentially therapeutic proteins that cannot be functionally expressed in bacteria. PROPOSED COMMERCIAL APPLICATIONS: There has been considerable interest in engineering human proteins for therapeutic use and directed evolution is the most efficient method for engineering many of these proteins due to the lack of structural information. Establishment of a mammalian host cell line for the expression of combinatorial protein libraries will enable the optimization of a broader range of potentially therapeutic proteins, particularly those that are not expressed functionally in bacteria.